Infection with Venezuelan equine encephalitis (VEE) virus causes an incapacitating febrile illness in people. Two vaccines (TC-83 and C-84) are currently available for human use as Investigational New Drugs, but both have limitations that prompted the development of an improved vaccine candidate. Unlike TC-83 and C-84, this new vaccine candidate (V3526) has a deletion of the PE2 glycoprotein cleavage site and a suppressor mutation at E1-253 (Davis et al., 1995, Virology 212: 102-110.).
Alphaviruses have protein spikes on their envelope which function during attachment of the virus to a cell. Three E1:E2 heterodimers associate to form one VEE virus glycoprotein spike. Normally, the E2 glycoprotein is cleaved at a furin-sensitive RKRR sequence from a PE2 precursor composed of E2 and E3 combined. Cleavage is believed to occur in the golgi or at the cell membrane, resulting in the production of E2 and E3 glycoproteins. The E3 glycoprotein is not present in the mature VEE virion. However, the deletion of the PE2 cleavage site in the V3526 candidate results in mature virions with spikes containing heterodimers of PE2 (without the RKRR cleavage sequence) and E1. The presence of the E3 protein in the spike could theoretically have profound implications on the ability of this virus to serve as a vaccine, by obscuring or altering the conformation of protective epitopes on the virus. However, animal studies (Davis et al., 1995, supra; Hart et. al., 2000, Vaccine 18, 3067-3075) indicate that V3526 induces protective immunity, such that V3526 remains a potential vaccine candidate. Alternatively, the inclusion of E3 in the spike could provide the immune system greater access to epitopes on this protein that would normally only be present as soluble protein either after PE2 cleavage or when infected cells lyse.
In order to identify potentially important epitopes on the E3 glycoprotein, monoclonal antibodies to V3526 were generated in 1994. Hybridoma supernatants were screened by ELISA for preferential binding to V3526 compared to VEE virus that cleaves the E3 protein (VEE Trinidad, TrD). Most of the 138 supernatants reacted equally to V3526 and TrD in ELISA and were not further evaluated. Six hybridomas were cloned twice and characterized. Surprisingly, the Mabs produced from these hybridomas, 13D4, 5E2, 5E4, 10D6, 10D7 and 3F2, notably 13D4, were found to protect mice from a lethal challenge with Venezuelan equine encephalitis (VEE) virus. The finding that Mabs with this specificity are protective is novel in that the E3 protein is normally cleaved from its precursor in infected cells and is not associated with most mature alphaviruses, and would not be expected to be a target of protective antibodies.
Therefore, this application describes protective VEE E3-specific MAbs. The antibodies are classified into one competition group based on competitive binding assays. One monoclonal antibody, namely, 13D4 protected BALB/c mice from death after challenge with virulent VEE virus when at least 20 ug of MAb was administered to the mice prior to challenge. The other five MAbs generally protected fewer than half of the recipient mice, even when a 50 ug dose of MAb was administered, although the mean time to death was extended significantly in these mice relative to control mice. The MAbs of the present invention may be used as a prophylactic or therapeutic treatment for alphavirus infections in other animal species, or in people. In addition, these Mabs may be used to distinguish in vitro between alphaviruses that have cleaved E3 and those that have not. This could be especially useful for diagnostic assays or in assays where the virus with E3 needs to be specifically neutralized.
Therefore, it is an object of the present invention to provide monoclonal antibodies which protect against VEE virus and bind to epitopes on the VEE virus E3 protein. Such antibodies are, for instance, produced by the cell line VE-V 13D4-1-1 deposited under the Budapest Treaty at American Type Culture Collection, Manassas, Va. on Apr. 14, 2002, having ATCC accession no. PTA-4248, and hybridoma cell line VE-V 5E2-1-1 deposited on Feb. 12, 2004, having ATCC accession no. PTA-5813, and hybridoma cell line VE-V 10D6-1-1 deposited on Feb. 12, 2004, having ATCC accession no. PTA-5814.
It is another object of the invention to provide for antibodies that are functionally equivalent to the antibodies listed above. These functionally equivalent antibodies substantially share at least one major functional property with an antibody listed above and herein described comprising: binding specificity to E3, protection against VEE challenge when administered prophylactically or therapeutically, or competition for the same binding site on E3. The antibodies can be of any class such as IgG, IgM, or IgA or any subclass such as IgG1, IgG2a, and other subclasses known in the art. Further, the antibodies can be produced by any method, such as phage display, or produced in any organism or cell line, including bacteria, plant, insect, mammal or other type of cell or cell line which produces antibodies with desired characteristics, such as humanized antibodies. The antibodies can also be formed by combining an Fab portion and a Fc region from different species.
The monoclonal antibodies of the present invention described below recognize epitopes on E3. The immunogen was the vaccine virus V3526 (Davis et al., 1995, Virology 212, 102-110; Hart et al., 2000. Vaccine 18, 3067-3075; Hart et al., 2002, Vaccine 20, 616-22; Steele et al., 1998, Vet. Pathl. 35, 386-97).
The sequence of the E3 protein which is not processed into E2, i.e. which do not contain a cleavage site, is presented below for different alphaviruses. These sequences/peptides can be used as immunogens for the production of protective antibodies, polyclonal or monoclonal, which recognize E3 of the different alphaviruses listed. The method for generating antibodies using peptides is well known in the art.
It is another object of the present invention to provide for mixtures of antibodies according to the present invention, as well as to methods of using individual antibodies, or mixtures thereof for the prevention and/or therapeutic treatment of VEE infections in vitro and in vivo, and/or for improved detection of VEE infections.
It is yet another object of the present invention to treat or prevent VEE virus infection by administering a therapeutically or prophylactically effective amount of one antibody of the present invention or a mixture of antibodies of the present invention to a subject in need of such treatment.
It is another object of the present invention to provide passive vaccines for treating or preventing VEE virus infections comprising a therapeutically or prophylactically effective amount of the antibodies of the present invention which protect against VEE virus, in combination with a pharmaceutically acceptable carrier or excipient. In a related aspect, similar antibodies generated from other alphaviruses can be similarly used as passive vaccines for treating or preventing such alphavirus infections.
It is still another object of the present invention to provide novel immunoprobes and test kits for detection of VEE virus which does not cleave its PE2 precursor, i.e. retains the E3 protein. For immunoprobes, the antibodies are directly or indirectly attached to a suitable reporter molecule, e.g., and enzyme or a radionuclide. The test kit includes a container holding one or more antibodies according to the present invention and instructions for using the antibodies for the purpose of binding to said VEE virus to form an immunological complex and detecting the formation of the immunological complex such that presence or absence of the immunological complex correlates with presence or absence of said VEE virus.
It is another object of the present invention to provide anti-idiotypic antibodies raised against one of the present monoclonal antibodies for use as a vaccine to elicit an active anti-E3 response.
It is yet another object of the present invention to provide antigenic epitopes as a component of a alphavirus vaccine. The epitopes described above comprising SEQ ID NO:1-5, or conservative changes thereof which are still recognized by the antibodies, are useful for actively immunizing a host to elicit production of protective antibodies against alphaviruses.